Ewing Sarcoma, a cancer of bone and soft tissue in children and young adults, is an aggressive malignancy with a poor long-term outcome. Even with optimal multi-agent chemotherapy, overall survival remains around 50%, drops to 25% for patients presenting with metastatic disease, and plummets to 10% for patients with recurrence. Thus, there is a tremendous need for better understanding of the biology of this disease, and the development of new therapies. The pathogenesis of Ewing Sarcoma is driven by EWS/Ets fusion oncoproteins. EWS/Ets oncoproteins, of which EWS/Fli1 is the most common, are aberrant transcription factors, which are necessary for Ewing Sarcoma oncogenesis. Although EWS/Ets fusion oncoproteins represent logical therapeutic targets in Ewing Sarcoma, such targeting has proven very difficult. Targeting of key downstream mediators of EWS/Ets oncogenic action is an important alternative strategy. We have identified an epigenetic regulator, the H3K9me1/2 histone demethylase KDM3A, which is overexpressed in Ewing Sarcoma downstream of EWS/Fli1, and promotes tumorigenesis and metastasis. Using transcriptome profiling and functional analysis, we have also identified candidate mediators of KDM3A action in Ewing Sarcoma. KDM3A is an enzyme belonging to the Jumonji-domain histone demethylase (JHDM) family, for which a small molecule inhibitor with in vivo efficacy has recently been developed, and shown by us to have activity against Ewing Sarcoma cells in vitro. Furthermore, our preliminary data identify at least one candidate mediator of KDM3A action with therapeutic targeting potential. The goal of this proposal is to gain better understanding of the precise roles and mechanism of action of KDM3A in Ewing Sarcoma, in order to determine the optimal strategies for inhibiting its pro-tumorigenic and pro-metastatic functions, and to test the efficacy of JHDM pharmacologic inhibition against these effects, as part of a long-term plan to develop new treatments for Ewing Sarcoma.